Laboratory and clinical procedures involving biospecific affinity procedures have dramatically affected health care and biological research. Such procedures, which are sometimes referred to as biospecific affinity reactions, are commonly employed in testing biological samples, such as blood or urine, for the identification, quantification, or both of a wide range of target substances. Biospecific affinity reactions have been used, for example, to identify particular chemical substances which have been correlated or associated with disease conditions. Biological entities such as proteins, biomolecules, nucleic acid sequences, e.g., mRNA, and the like, are preferred "target substances" or "target particles" as those terms are used herein.
Various methods are available for identifying the presence or quantity of the above-mentioned target substances in a given medium or environment based upon formation of a complex or a binding reaction between the target substance, i.e., a ligand of interest, and a specific binding partner. The binding partner preferentially binds to the ligand and not to other constituents which may be present in the sample. In each instance, the occurrence or degree of target substance/binding partner complex formation is determinable.
Assays typically used in the art include immunoassays, hybridization assays, and protein-ligand assays. Immunoassays are based upon the specificity of an antibody for an antigen. Hybridization assays are based upon the specificity of complementary nucleic acid sequences, i.e., on the hybridization of nucleic acid probes, with target nucleic acids. Protein-ligand assays depend upon the affinity of a binding site on a protein for a specific ligand, e.g., streptavidin for biotin.
In each assay type, quantitation of the target substance requires a physical separation of bound from free unlabeled ligand or receptor.
In one approach, physical separation of bound from free ligand or receptor may be accomplished gravitationally, e.g., by settling, or, alternatively, by centrifugation, of small diameter particles or beads coupled to the target substance. If desired, such particles or beads may be or can be made magnetic to facilitate the bound/free separation step. Magnetic particles are well known in the art, as is their use in immune and other bio-specific affinity reactions. See, for example, U.S. Pat. No. 4,554,088 and Immunoassays for Clinical Chemistry, pp. 147-162, Hunter et al., eds., Churchill Livingston, Edinborough (1983). Generally, any material which facilitates magnetic separation may be employed for this purpose.
Small diameter or small dimensioned magnetic particles have proven to be quite useful in analyses involving biospecific affinity reactions, as they can be conveniently coated with biofunctional molecules, e.g., proteins, which have a binding site for a specific ligand and provide favorable reaction kinetics. Magnetic particles ranging in longest dimension from 3 nm to many microns (and larger) have been described in the patent literature, including, by way of example and not by way of limitation, U.S. Pat. Nos. 3,970,518; 4,018,886; 4,230,685; 4,267,234; 4,452,773; 4,554,088; 4,659,678; 4,978,610; and 5,200,084.
Smaller sized magnetic particles, such as those mentioned above, generally fall into two broad categories. The first category includes particles that are permanently magnetized; and the second comprises particles that become magnetic only when subjected to a magnetic field. Permanently magnetized particles are often referred to as "ferromagnetic". The second class of particles are generally referred to as paramagnetic. For purposes of this application, both classes of particles are collectively referred to herein as "magnetically responsive" particles.
The type of magnetic separation device used for separating target substance-bearing particles from test media will depend on the chemical nature and the particle size of the magnetic particle utilized. Micron-sized paramagnetic particles are readily removed from surrounding media, e.g., a solution, by means of commercially available magnetic separation devices, employing relatively inexpensive permanent magnets. Examples of such magnetic separators are the MAIA Magnetic Separator manufactured by Serono Diagnostics, Norwell, Mass., U.S.A., the DYNAL MPC-1 manufactured by DYNAL, Inc., Great Neck, N.Y., U.S.A., and the BioMag Separator, manufactured by Advanced Magnetics, Inc., Cambridge, Mass., U.S.A. A similar magnetic separator, manufactured by Ciba-Corning Medical Diagnostics, Wampole, Mass., U.S.A., is provided with rows of bar magnets arranged in parallel and located at the base of the separator. The Ciba-Corning device accommodates 60 test tubes, with the closed end of each tube fitting into a recess between two of the bar magnets.
For separation of particles of smaller size, e.g., colloidal dimension and smaller, higher field strength, e.g., higher gradient field separators, may be used. The present invention can be used in the separation of target particles from a medium including such particles, regardless of particle size. The present invention also is applicable to separate target particle/ligand complexes from a medium where the target particle/ligand complex is removable from the medium by techniques other than magnetic. In essence, this invention can be used in any circumstance where a complex is created in a medium for the purpose of isolation or identification and there are substances or chemical species present in the medium which tend to interfere with the formation of the ligand/receptor complex or target particle/binding agent complex or which tend to promote complex breakdown.
In a broader sense, aspects of this invention are applicable to any situation where a suspension or a solution of a target material is to be physically separated from an interfering substance or substances which can be collected or localized, e.g., by centrifugation. This invention provides the means by which the step of separating a target material from an interfering substance, e.g., by transfer of one away from the other via pipette or other means, can be completely avoided.
The concomitant of this invention is that where interfering substances can be localized, e.g., by centrifugation, subsequent processing of the target particles or target substance solution/suspension can be accomplished in the same centrifuge sample well.
This invention results in savings of time, e.g., by eliminating transfer steps, and minimizes the quantity of expensive or potentially scarce or difficult to obtain starting materials. This invention reduces the likelihood of sample cross contamination, reduces user error, provides greater reproductability of results, and provides a significant reduction in the use of disposable pieces, e.g., micropipette tips and tubes. Reproducibility can be obtained even though very small samples of biological interest, e.g., 20 mg or less, are used. Where, for example, analysis of tissue or organ samples obtained in a biopsy, is contemplated, the ability to do reproducible, qualitative or quantitative analyses on small, biological samples can be very important to reduce patient trauma. Lastly, as is described below, this invention is particularly applicable to automated processing systems and can be used to enhance their efficiency.
This invention solves a particular problem which sometimes occurs during the formation of complexes such as receptor/ligand complexes or in the separation of such receptor/ligand complexes from the medium in which they are dispersed. Specifically, it was found that, in the isolation of mRNA, after cell disruption or homogenation, dilution and centrifugation to generate cleared cell lysates, the precipitated proteins and cellular debris in the end of the centrifuge tube (usually in the form of a pellet) would interfere with complex formation between magnetic streptavidin particles and biotinylated oligo (dT):mRNA complexes. The specific interference caused by the unwanted proteins and cellular debris was interference in the release of mRNA from magnetic streptavidin particles upon addition of distilled water to collected streptavidin particles having biotinylated oligo (dT):mRNA complexes thereon.
One approach to this problem was simply to transfer cleared lysate from the centrifuge tube into a sterile tube and to perform the mRNA purification procedure, e.g., by formation of a magnetic complex, in the absence of cell and tissue debris. This approach generally involved pipetting of cleared lysate from one tube to another. Where a limited number of samples was utilized, e.g., five or less, physical transference of cleared lysate by pipetting was and is an acceptable procedure.
However, in the context of, for example, high throughput, multiple-well (e.g., five or more) plate magnetic isolation devices, performing a physical transfer step, such as pipetting, is time consuming, prone to error, subject to sample contamination or sample loss, generates large quantities of unwanted disposables, e.g., plastic pipette tubes, and permits the undesirable transfer of interfering substances.
The present invention therefore is applicable in the circumstance where target substances are to be isolated from a medium using multiple well plates and interference with complex formation or isolation, as discussed above, is experienced. The invention provides enhanced reproducibility, even where small or very small samples are involved. A specific, preferred, multiple well plate used in a magnetic separation process is that described in applicants' commonly assigned and concurrently filed patent application referred to above.